Antibody variants

ABSTRACT

Antibody variants of parent antibodies are disclosed which have one or more amino acids inserted in a hypervariable region of the parent antibody and a binding affinity for a target antigen which is at least about two fold stronger than the binding affinity of the parent antibody for the antigen.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation application claiming priority to U.S. applicationSer. No. 15/384,962, filed Dec. 20, 2016, which is a continuationapplication of U.S. application Ser. No. 14/743,933, filed Jun. 18, 2015(now abandoned), which is a continuation application of U.S. applicationSer. No. 14/529,569, filed Oct. 31, 2014 (now abandoned), which is acontinuation application of U.S. application Ser. No. 14/195,190, filedMar. 3, 2014 (now abandoned), which is a continuation application ofU.S. application Ser. No. 13/052,982, filed Mar. 21, 2011, now U.S. Pat.No. 8,703,133, issued Apr. 22, 2014, which is a continuation applicationof U.S. application Ser. No. 12/402,374, filed Mar. 11, 2009 (nowabandoned), which is a continuation application of U.S. application Ser.No. 11/552,445, filed Oct. 24, 2006 (now abandoned), which is acontinuation application of U.S. application Ser. No. 10/624,153, filedJul. 21, 2003 (now abandoned), which is a continuation application ofU.S. application Ser. No. 09/440,781, filed Nov. 16, 1999, now U.S. Pat.No. 6,632,926, issued Oct. 14, 2003, which is a non-provisionalapplication filed under 37 CFR 1.53(b) claiming priority to provisionalapplication 60/108,945 filed Nov. 18, 1998, the contents of which areincorporated herein by their references.

SEQUENCE LISTING

This application contains a Sequence Listing submitted via EFS-Web andhereby incorporated by reference in its entirety. Said ASCII copy,created on Jul. 3, 2018, is named P01469-US-11SequenceListing.txt, andis 40,796 bytes in size.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates generally to antibody variants. In particular,antibody variants of parent antibodies are disclosed which have one ormore amino acids inserted in a hypervariable region of the parentantibody and a binding affinity for a target antigen which is at leastabout two fold stronger than the binding affinity of the parent antibodyfor the antigen.

Description of Related Art

Antibodies are proteins, which exhibit binding specificity to a specificantigen. Native antibodies are usually heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two identical light (L) chains andtwo identical heavy (H) chains. Each light chain is linked to a heavychain by one covalent disulfide bond, while the number of disulfidelinkages varies between the heavy chains of different immunoglobulinisotypes. Each heavy and light chain also has regularly spacedintrachain disulfide bridges. Each heavy chain has at one end a variabledomain (V_(H)) followed by a number of constant domains. Each lightchain has a variable domain at one end (V_(L)) and a constant domain atits other end; the constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areresponsible for the binding specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in three segments called Complementarity DeterminingRegions (CDRs) both in the light chain and the heavy chain variabledomains. The more highly conserved portions of the variable domains arecalled the framework regions (FR). The variable domains of native heavyand light chains each comprise four FR regions, largely adopting aβ-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the β-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)).

The constant domains are not involved directly in binding an antibody toan antigen, but exhibit various effector functions. Depending on theamino acid sequence of the constant region of their heavy chains,antibodies or immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG andIgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. The heavychain constant regions that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. Of thevarious human immunoglobulin classes, only human IgG1, IgG2, IgG3 andIgM are known to activate complement.

In vivo, affinity maturation of antibodies is driven by antigenselection of higher affinity antibody variants which are made primarilyby somatic hypermutagenesis. A “repertoire shift” also often occurs inwhich the predominant germline genes of the secondary or tertiaryresponse are seen to differ from those of the primary or secondaryresponse.

Various research groups have attempted to mimic the affinity maturationprocess of the immune system, by introducing mutations into antibodygenes in vitro and using affinity selection to isolate mutants withimproved affinity. Such mutant antibodies can be displayed on thesurface of filamentous bacteriophage and antibodies can be selected bytheir affinity for antigen or by their kinetics of dissociation(off-rate) from antigen. Hawkins et al. J. Mol. Biol. 226:889-896(1992). CDR walking mutagenesis has been employed to affinity maturehuman antibodies which bind the human envelope glycoprotein gp120 ofhuman immunodeficiency virus type 1 (HIV-1) (Barbas III et al. PNAS(USA) 91: 3809-3813 (1994); and Yang et al. J. Mol. Biol. 254:392-403(1995)); and an anti-c-erbB-2 single chain Fv fragment (Schier et al. J.Mol. Biol. 263:551567 (1996)). Antibody chain shuffling and CDRmutagenesis were used to affinity mature a high-affinity human antibodydirected against the third hypervariable loop of HIV (Thompson et al. J.Mol. Biol. 256:77-88 (1996)). Balint and Larrick Gene 137:109-118 (1993)describe a technique they coin “parsimonious mutagenesis” which involvescomputer-assisted oligodeoxyribonucleotide-directed scanning mutagenesiswhereby all three CDRs of a variable region gene are simultaneously andthoroughly searched for improved variants. Wu et al. affinity matured anαvβ3-specific humanized antibody using an initial limited mutagenesisstrategy in which every position of all six CDRs was mutated followed bythe expression and screening of a combinatorial library including thehighest affinity mutants (Wu et al. PNAS (USA) 95: 6037-6-42 (1998)).Phage antibodies are reviewed in Chiswell and McCafferty TIBTECH10:80-84 (1992); and Rader and Barbas III Current Opinion in Biotech.8:503-508 (1997). In each case where mutant antibodies with improvedaffinity compared to a parent antibody are reported in the abovereferences, the mutant antibody has amino acid substitutions in a CDR.

SUMMARY OF THE INVENTION

Unlike the affinity matured antibodies of the above references, thepresent invention provides an antibody variant of a parent antibody,which antibody variant comprises an amino acid insertion in or adjacentto a hypervariable region of the parent antibody and has a bindingaffinity for a target antigen which is at least about two fold strongerthan the binding affinity of the parent antibody for the antigen.

The invention further provides an antibody variant comprising a heavychain variable domain, wherein CDR H3 of the heavy chain variable domaincomprises the amino acid sequence of CDR H3 of a variant selected fromthe group consisting of Y0239-19 (SEQ ID NO:85); Y0239-8 (SEQ ID NO:53);Y0240-1 (SEQ ID NO:86); Y0239-12 (SEQ ID NO:78); Y0239-9 (SEQ ID NO:54);and Y0261-6 (SEQ ID NO:89). These CDR H3 sequences may, for example, beprovided in the heavy chain variable domain sequence of SEQ ID NO: 98 or99; see FIG. 1B). Preferably, the antibody variant further comprises alight chain variable domain and binds VEGF antigen with stronger bindingaffinity than Y0192 (see FIGS. 1A and 1B; SEQ ID NO's 95 and 96).

The invention further provides a method for producing an antibodyvariant comprising introducing an amino acid residue in or adjacent to ahypervariable region of a parent antibody, wherein the antibody varianthas a binding affinity for a target antigen which is at least about twofold stronger than the binding affinity of the parent antibody for saidantigen.

Additionally, the invention provides a method for making an antibodyvariant, comprising the steps of:

(a) identifying potential amino acid interactions between ahypervariable region of a parent antibody and a target antigen;

(b) preparing a variant of the parent antibody comprising introducing anamino acid residue in or adjacent to the hypervariable region of theparent antibody, wherein the introduced amino acid residue contributesto the potential amino acid interactions in (a); and

(c) selecting an antibody variant prepared as in (b) which has astronger binding affinity for the antigen than the parent antibody.

Various forms of the antibody variant are contemplated herein. Forexample, the antibody variant may be a full length antibody (e.g. havinga human immunoglobulin constant region) or an antibody fragment (e.g. aF(ab′)₂). Furthermore, the antibody variant may be labeled with adetectable label, immobilized on a solid phase and/or conjugated with aheterologous compound (such as a cytotoxic agent).

Diagnostic and therapeutic uses for the antibody variant arecontemplated. In one diagnostic application, the invention provides amethod for determining the presence of an antigen of interest comprisingexposing a sample suspected of containing the antigen to the antibodyvariant and determining binding of the antibody variant to the sample.For this use, the invention provides a kit comprising the antibodyvariant and instructions for using the antibody variant to detect theantigen.

The invention further provides: isolated nucleic acid encoding theantibody variant; a vector comprising the nucleic acid, optionally,operably linked to control sequences recognized by a host celltransformed with the vector; a host cell transformed with the vector; aprocess for producing the antibody variant comprising culturing thishost cell so that the nucleic acid is expressed and, optionally,recovering the antibody variant from the host cell culture (e.g. fromthe host cell culture medium).

The invention also provides a composition comprising the antibodyvariant and a pharmaceutically acceptable carrier or diluent. Thiscomposition for therapeutic use is sterile and may be lyophilized.

The invention further provides a method for treating a mammal comprisingadministering an effective amount of the antibody variant to the mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show a sequence alignment of the light chain variableregion (FIG. 1A) and heavy chain variable region (FIG. 1B) of severalvariants of the humanized anti-VEGF antibody F(ab)-12. The parentalFab-phage clone Y0192 contains light chain mutations which do notsignificantly affect antigen binding affinity, and has been described(WO98/45331). Another variant, Y0238-3, contains mutations in CDR H1which improve antigen-binding (WO98/45331). Variant Y0239-19 containsthe “VNERK” motif (SEQ ID NO: 100) identified in selections from CDR H3insertion libraries described herein. Variant Y0313-2 contains the CDRH1 mutations of Y0238-3 combined with the CDR H3 mutations of Y0239-19.Differences from F(ab)-12 are highlighted with shaded boxes. Thesequence identifiers in FIGS. 1A and 1B are as follows: F(ab)-12 lightchain variable domain (SEQ ID NO:94); Y0192, Y0238-3, Y0239-19 andY0313-2 light chain variable domain (SEQ ID NO:95); F(ab)-12 and Y0192heavy chain variable domain (SEQ ID NO:96); Y0238-3 heavy chain variabledomain (SEQ ID NO:97); Y0239-19 heavy chain variable domain (SEQ IDNO:98); and Y0313-2 heavy chain variable domain (SEQ ID NO:99).

FIG. 2 shows the inhibition of VEGF activity in a cell-based bioassay byFab, F(ab)-12 and Fab variant Y0313-2.

FIG. 3 shows a portion of the three-dimensional model of F(ab)-12 incomplex with VEGF as determined by x-ray crystallography (Muller et al.Structure 6(9): 1153-1167 (1998)). The main chain trace of the CDR H3region of the antibody is depicted as a magenta ribbon at right. Asurface rendering of a portion of VEGF is depicted at left, with severalproximal residues highlighted in red (acidic) or purple (basic). Theside chain of D41 of VEGF can be seen as a site of potential interactionwith a hypothetical insertion peptide placed into the CDR H3.

FIG. 4 shows a superposition of portions of the three-dimensional modelof F(ab)-12 in complex with VEGF (both molecules in gray; Muller et al.,supra) with a model of the insertion variant Fab Y0313-2 (green) incomplex with VEGF (yellow). The latter model is based on x-raycrystallographic determination of the variant complex structuredescribed herein. The figure illustrates that little structural changeis observed in the complex as compared with the F(ab)-12 complex, exceptin the immediate vicinity of the mutations V104, N104a, E104b, R104c,and K105.

FIG. 5 shows a comparison of portions of the three-dimensional model ofF(ab)-12 in complex with VEGF (at right; Muller et al., supra) with amodel of Fab Y0313-2 in complex with VEGF (at left) as described herein.In each case, VEGF is shown in yellow, and the respective Fab is shownin green. In the Y0313-2 complex, it can be seen that V104 and R104cmake new contacts with VEGF.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity.

The term “hypervariable region” when used herein refers to the regionsof an antibody variable domain which are hypervariable in sequenceand/or form structurally defined loops. The hypervariable regioncomprises amino acid residues from a “complementarity determiningregion” or “CDR” (i.e. residues 24-34 (“CDR L1”), 50-56 (“CDR L2”) and89-97 (“CDR L3”) in the light chain variable domain and 31-35 (“CDRH1”), 50-65 (“CDR H2”) and 95-102 (“CDR H3”) in the heavy chain variabledomain; Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991)) and/or those residues from a “hypervariable loop” (i.e.residues 26-32 (“loop L1”), 50-52 (“loop L2”) and 91-96 (“loop L3”) inthe light chain variable domain and 26-32 (“loop H1”), 53-55 (“loop H2”)and 96-101 (“loop H3”) in the heavy chain variable domain; Chothia andLesk J. Mol. Biol. 196:901-917 (1987)). In both cases, the variabledomain residues are numbered according to Kabat et al., supra.“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

The expression “variable domain residue numbering as in Kabat” refers tothe numbering system used for heavy chain variable domains or lightchain variable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of CDR H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol.Biol. 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues which are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

The expression “linear antibodies” when used throughout this applicationrefers to the antibodies described in Zapata et al. Protein Eng.8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair oftandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

A “parent antibody” is an antibody comprising an amino acid sequencewhich lacks, or is deficient in, one or more amino acid residues in oradjacent to one or more hypervariable regions thereof compared to anantibody variant as herein disclosed. Thus, the parent antibody has ashorter hypervariable region than the corresponding hypervariable regionof an antibody variant as herein disclosed. The parent polypeptide maycomprise a native sequence (i.e. a naturally occurring) antibody(including a naturally-occurring allelic variant) or an antibody withpre-existing amino acid sequence modifications (such as otherinsertions, deletions and/or substitutions) of a naturally-occurringsequence. Preferably the parent antibody is a humanized antibody or ahuman antibody.

As used herein, “antibody variant” refers to an antibody which has anamino acid sequence which differs from the amino acid sequence of aparent antibody. Preferably, the antibody variant comprises a heavychain variable domain or a light chain variable domain having an aminoacid sequence which is not found in nature. Such variants necessarilyhave less than 100% sequence identity or similarity with the parentantibody. In a preferred embodiment, the antibody variant will have anamino acid sequence from about 75% to less than 100% amino acid sequenceidentity or similarity with the amino acid sequence of either the heavyor light chain variable domain of the parent antibody, more preferablyfrom about 80% to less than 100%, more preferably from about 85% to lessthan 100%, more preferably from about 90% to less than 100%, and mostpreferably from about 95% to less than 100%. Identity or similarity withrespect to this sequence is defined herein as the percentage of aminoacid residues in the candidate sequence that are identical (i.e sameresidue) with the parent antibody residues, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity. None of N-terminal, C-terminal, or internalextensions, deletions, or insertions into the antibody sequence outsideof the variable domain shall be construed as affecting sequence identityor similarity. The antibody variant is generally one which has a longerhypervariable region (by one or more amino acid residues; e.g. by aboutone to about 30 amino acid residues and preferably by about two to aboutten amino acid residues) than the corresponding hypervariable region ofa parent antibody.

An “amino acid alteration” refers to a change in the amino acid sequenceof a predetermined amino acid sequence. Exemplary alterations includeinsertions, substitutions and deletions.

An “amino acid insertion” refers to the introduction of one or moreamino acid residues into a predetermined amino acid sequence

The amino acid insertion may comprise a “peptide insertion” in whichcase a peptide comprising two or more amino acid residues joined bypeptide bond(s) is introduced into the predetermined amino acidsequence. Where the amino acid insertion involves insertion of apeptide, the inserted peptide may be generated by random mutagenesissuch that it has an amino acid sequence which does not exist in nature.

The inserted residue or residues may be “naturally occurring amino acidresidues” (i.e. encoded by the genetic code) and selected from the groupconsisting of: alanine (Ala); arginine (Arg); asparagine (Asn); asparticacid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu);glycine (Gly); histidine (His); isoleucine (Ile): leucine (Leu); lysine(Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine(Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine(Val).

Insertion of one or more non-naturally occurring amino acid residues isalso encompassed by the definition of an amino acid insertion herein. A“non-naturally occurring amino acid residue” refers to a residue, otherthan those naturally occurring amino acid residues listed above, whichis able to covalently bind adjacent amino acid residues(s) in apolypeptide chain. Examples of non-naturally occurring amino acidresidues include norleucine, ornithine, norvaline, homoserine and otheramino acid residue analogues such as those described in Ellman et al.Meth. Enzym. 202:301-336 (1991). To generate such non-naturallyoccurring amino acid residues, the procedures of Noren et al. Science244:182 (1989) and Ellman et al., supra, can be used. Briefly, theseprocedures involve chemically activating a suppressor tRNA with anon-naturally occurring amino acid residue followed by in vitrotranscription and translation of the RNA.

An amino acid insertion “in a hypervariable region” refers to theintroduction of one or more amino acid residues within a hypervariableregion amino acid sequence.

An amino acid insertion “adjacent a hypervariable region” refers to theintroduction of one or more amino acid residues at the N-terminal and/orC-terminal end of a hypervariable region, such that at least one of theinserted amino acid residues forms a peptide bond with the N-terminal orC-terminal amino acid residue of the hypervariable region in question.

An “amino acid substitution” refers to the replacement of an existingamino acid residue in a predetermined amino acid sequence with anotherdifferent amino acid residue.

The term “potential amino acid interactions” refers to contacts orenergetically favorable interactions between one or more amino acidresidues present in an antigen and one or more amino acid residues whichdo not exist in a parent antibody but can be introduced therein so as toincrease the amino acid contacts between the antigen and an antibodyvariant comprising those introduced amino acid residue(s). Preferablythe the amino acid interactions of interest are selected from the groupconsisting of hydrogen bonding, van der Waals interactions and ionicinteractions

The term “target antigen” herein refers to a predetermined antigen towhich both a parent antibody and antibody variant as herein definedbind. The target antigen may be polypeptide, carbohydrate, nucleic acid,lipid, hapten or other naturally occurring or synthetic compound.Preferrably, the target antigen is a polypeptide. While the antibodyvariant binds the target antigen with better binding affinity than theparent antibody, the parent antibody generally has a binding affinity(K_(d)) value for the target antigen of no more than about 1×10⁻⁵M, andpreferably no more than about 1×10⁻⁶M.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

A “disorder” is any condition that would benefit from treatment with theantibody variant. This includes chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, nonhuman primates,and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeAdriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside (“Ara-C”),Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin,Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin,Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine,Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin,Aminopterin, Dactinomycin, Mitomycins, Esperamicins (see U.S. Pat. No.4,675,187), Melphalan and other related nitrogen mustards.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,beta-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibody.The label may be itself be detectable (e.g., radioisotope labels orfluorescent labels) or, in the case of an enzymatic label, may catalyzechemical alteration of a substrate compound or composition which isdetectable.

By “solid phase” is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g. an affinity chromatography column). This term also includesa discontinuous solid phase of discrete particles, such as thosedescribed in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as the antibody variants disclosed herein and, optionally, achemotherapeutic agent) to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

II. Modes for Carrying Out the Invention

The invention herein relates to a method for making an antibody variant.The parent antibody or starting antibody is prepared using techniquesavailable in the art for generating such antibodies. Exemplary methodsfor generating antibodies are described in more detail in the followingsections.

The parent antibody is directed against a target antigen of interest.Preferably, the target antigen is a biologically important polypeptideand administration of the antibody to a mammal suffering from a diseaseor disorder can result in a therapeutic benefit in that mammal. However,antibodies directed against nonpolypeptide antigens (such astumor-associated glycolipid antigens; see U.S. Pat. No. 5,091,178) arealso contemplated.

Where the antigen is a polypeptide, it may be a transmembrane molecule(e.g. receptor) or ligand such as a growth factor. Exemplary antigensinclude molecules such as renin; a growth hormone, including humangrowth hormone and bovine growth hormone; growth hormone releasingfactor; parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; proteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor; platelet-derived growth factor(PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growthfactor (EGF); transforming growth factor (TGF) such as TGF-alpha andTGF-beta; insulin-like growth factor-I and -II (IGF-I and IGF-II);des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor bindingproteins; CD proteins such as CD3, CD4, CD8, CD19 and CD20;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CDlla, CDllb, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments of any of the above-listed polypeptides.

Preferred molecular targets for antibodies encompassed by the presentinvention include CD proteins such as CD3, CD4, CD8, CD19, CD20 andCD34; members of the ErbB receptor family such as the EGF receptor,HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1,Mac1, p150, 95, VLA-4, ICAM-1, VCAM and αv/β3 integrin including eitheralpha or beta subunits thereof (e.g. anti-CD11a, anti-CD18 or anti-CD11bantibodies); growth factors such as VEGF; IgE; blood group antigens;flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; proteinC etc.

The antigen used to generate an antibody may be isolated from a naturalsource thereof, or may be produced recombinantly or made using othersynthetic methods. Alternatively, cells comprising native or recombinantantigen can be used as immunogens for making antibodies.

The parent antibody may have pre-existing strong binding affinity forthe target antigen. For example, the parent antibody may bind theantigen of interest with a binding affinity (K_(d)) value of no morethan about 1×10⁻⁷ M, preferably no more than about 1×10⁻⁸ M and mostpreferably no more than about 1×10⁻⁹ M.

Antibody “binding affinity” may be determined by equilibrium methods(e.g. enzyme-linked immunoabsorbent assay (ELISA) or radioimmunoassay(RIA)), or kinetics (e.g. BIACORE™ analysis; see Example 1 below), forexample.

Also, the antibody may be subjected to other “biological activityassays”, e.g., in order to evaluate its “potency” or pharmacologicalactivity and potential efficacy as a therapeutic agent. Such assays areknown in the art and depend on the target antigen and intended use forthe antibody. Examples include the keratinocyte monolayer adhesion assayand the mixed lymphocyte response (MLR) assay for CD11a (seeWO98/23761); tumor cell growth inhibition assays (as described in WO89/06692, for example); antibody-dependent cellular cytotoxicity (ADCC)and complement-mediated cytotoxicity (CDC) assays (U.S. Pat. No.5,500,362); agonistic activity or hematopoiesis assays (see WO95/27062); tritiated thymidine incorporation assay; and alamar blueassay to measure metabolic activity of cells in response to a moleculesuch as VEGF (See Example 1 below).

The amino acid sequence of the parent antibody is altered so as togenerate an antibody variant which has a stronger binding affinity forthe target antigen than the parent antibody. The antibody variantpreferably has a binding affinity for the target antigen which is atleast about two fold stronger (e.g. from about two fold to about 1000fold or even to about 10,000 fold improved binding affinity), preferablyat least about five fold stronger, and preferably at least about tenfold or 100 fold stronger, than the binding affinity of the parentantibody for the antigen. The enhancement in binding affinity desired orrequired may depend on the initial binding affinity of the parentantibody.

Where the assay used is a biological activity assay, the antibodyvariant preferably has a potency in the biological activity assay ofchoice which is at least about two fold greater (e.g. from about twofold to about 1000 fold or even to about 10,000 fold improved potency),preferably at least about 20 fold greater, more preferably at leastabout 50 fold greater, and sometimes at least about 100 fold or 200 foldgreater, than the biological activity of the parent antibody in thatassay.

To generate the antibody variant, one or more amino acid residues areintroduced or inserted in or adjacent to one or more of thehypervariable regions of the parent antibody. Generally, one will insertone or more amino acid residues in a CDR of the parent antibody. Thenumber of residues to be inserted may be from about one residue to about30 amino acid residues, e.g. from about two to about ten amino acidresidues. In deciding the number of residues to be inserted, one maytake into account the range of lengths of the hypervariable region inquestion in known antibodies. For example, for the first hypervariableregion of a light chain variable domain, the hypervariable region ispreferably “CDR L1” according to Kabat et al., supra, e.g. having anoverall length from about nine amino acid residues to about 20 residues,including the inserted amino acid residue(s). With respect to the secondhypervariable region of a light chain variable domain, the hypervariableregion is preferably “CDR L2” according to Kabat et al., supra, e.g.having an overall length from about five amino acid residues to aboutten residues, including the inserted amino acid residue(s). In relationto the third hypervariable region of a light chain variable domain, thehypervariable region is preferably “CDR L3” according to Kabat et al.,supra, e.g. having an overall length from about seven amino acidresidues to about 20 residues, including the inserted amino acidresidue(s).

Preferably, the antibody variant has one or more amino acid residuesinserted in a hypervariable region of the heavy chain variable region,most preferably CDR H3. If this hypervariable region is chosen,preferably the inserted amino acid residues are between residue numbers97 and 102 (e.g., adjacent to, and preferably C-terminal in sequence to,residue number 100) of the heavy chain variable domain of the parentantibody, utilizing the variable domain residue numbering as in Kabat.

In deciding upon the number of amino acid residues to insert, one maytake into account the desired length of the altered hypervariableregion. For example, for the first hypervariable region of a heavy chainvariable domain, the hypervariable region is preferably the stretch ofresidues from the “loop H1” of Chothia et al, supra, combined with thestretch of residues considered to constitute “CDR H1” according to Kabatet al., supra. Thus, this first hypervariable loop of the heavy chainvariable domain may have an overall length from about eight amino acidresidues to about 20 residues including the inserted amino acidresidue(s). In relation to the second hypervariable region of a heavychain variable domain, the hypervariable region is preferably “CDR H2”according to Kabat et al., supra, e.g. having an overall length fromabout 14 amino acid residues to about 25 residues, including theinserted amino acid residue(s). Finally, in relation to the thirdhypervariable region of a heavy chain variable domain, the hypervariableregion is preferably “CDR H3” according to Kabat et al., supra, e.g.having an overall length from about six amino acid residues to about 30residues, including the inserted amino acid residue(s).

Antibody variants with inserted amino acid residue(s) in a hypervariableregion thereof may be prepared randomly, especially where the startingbinding affinity of the parent antibody for the target antigen is suchthat randomly produced antibody variants can be readily screened. Forexample, phage display provides a convenient method of screening suchrandom variants.

The invention also provides a more systematic method for making antibodyvariants. This method involves the following general steps, usuallyperformed sequentially:

(a) identifying potential amino acid interactions between ahypervariable region of a parent antibody and a target antigen;

(b) preparing a variant of the parent antibody by introducing an aminoacid residue in or adjacent to the hypervariable region of the parentantibody, wherein the introduced amino acid residue contributes to thepotential amino acid interactions in (a); and

(c) selecting an antibody variant prepared as in (b) which has astronger binding affinity for the antigen than the parent antibody.

According to step (a) of this method, one may analyze a molecular modelof the parent antibody complexed with antigen. The molecular model maybe obtained from an X-ray crystal or nuclear magnetic resonance (NMR)structure of this complex. See, e.g., Amit et al. Science 233:747-753(1986); and Muller et al. Structure 6(9): 1153-1167 (1998)).Alternatively, computer programs can be used to create molecular modelsof antibody/antigen complexes (see, e.g., Levy et al. Biochemistry28:7168-7175 (1989); Bruccoleri et al. Nature 335: 564-568 (1998); andChothia et al. Science 233: 755-758 (1986)), where a crystal structureis not available.

In the preferred method, one analyzes the molecular model of theantigen/antibody complex and identifies potential areas for increasingenergetically favorable interactions between the antigen and ahypervariable region of the antibody. For example, one may identifypotential polar interactions (e.g. ion pairs and/or hydrogen-bonding);non-polar interactions (such as Van der Waals attractions and/orhydrophobic interactions); and/or covalent interactions (e.g. disulfidebond(s)) between one or more amino acid residues of the antigen and oneor more amino acid residues which can be inserted in or adjacent to ahypervariable region of the antibody. Preferably at least one of theinserted residues has a net positive charge or a net negative charge.For example, at least one of the inserted residues may be a positivelycharged residue, preferably arginine or lysine.

Examples of side chains typically having positive charge are lysine,arginine, and histidine. Examples of side chains typically havingnegative charge are aspartic acid and glutamic acid. These side chainsmay undergo ionic interactions (positive residues paired with negativeresidues), as well as polar interactions with side chains having polarfunctional groups: tryptophan, serine, threonine, tyrosine, cysteine,tyrosine, asparagine, and glutamine. In addition, polar or ionicinteractions may be mediated through intervening solvent (such as water)or solute (e.g. phosphate or sulfate) molecules.

Examples of residues which may be involved in hydrophobic interactions,or non-polar Van der Waals interactions, are typically alanine, valine,leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, andtyrosine; however, the non-polar side chains of other residues, such aslysine or arginine, may also participate in such interactions. Aromaticside chains such as phenylalanine, tyrosine, and tryptophan may formaromatic (pi) stacking interactions, or may act as hydrogen-bondacceptors.

In addition, the main chain atoms of any residue (including glycine) mayundergo Van der Waals or hydrophobic interactions; and the atomsnitrogen and carbonyl oxygen of the main chain, may undergo polar(hydrogen-bonding) interactions. In some cases, a covalent bond(disulfide) may be formed from a cysteine residue of the antibody with acysteine residue of the antigen.

Finally, post-translational modifications (e.g., glycosylation orphosphorylation) or a prosthetic group (e.g., heme or zinc finger) mayprovide additional functional groups (carboxylate or phosphate oxygens;zinc or iron atoms) for interaction between antibody and antigen.

Thus, one may, for example, introduce one or more charged amino acidresidues in or adjacent to a hypervariable region of the parent antibodyin an appropriate three dimensional location, such that the introducedresidue or residues are able to form ion pair(s) with one or moreoppositely charged residues in the antigen. Similarly, one can createhydrogen-bonding pair(s), Van der Waals interactions, etc., byintroducing appropriate amino acid residues in an appropriate locationin or adjacent to a hypervariable region of the antibody.

The antibody variant may comprise additional alterations, such as aminoacid deletions or substitutions in the hypervariable region of theantibody in which the insertion is made. This is shown in the examplebelow, wherein the hypervariable region was modified by both amino acidsubstitutions as well as amino acid insertions.

In general, any inserted amino acid residue or inserted peptide willneed to exit the existing antibody polypeptide chain at a residueposition (x), extend to a point sufficiently near to the site of a newcontact such that some portion of the amino acid side chain or mainchain of the peptide can form an interaction, and return to reenter theexisting antibody polypeptide chain at a position (y) (where y>x in thelinear sequence).

It is desirable that the inserted amino acid residue or peptide notsignificantly perturb the structure of the antibody in a global or localsense, beyond the vicinity of the newly inserted amino acid residue orpeptide. In particular, the inserted amino acid residue or peptidepreferably does not distort the FR residues of the antibody, or residuesof the antibody or antigen involved in existing contacts. This may beevaluated in an actual or modeled complex.

If both exit/reentry residues (x and y) lack significant intramolecularand intermolecular contacts (i.e., both within the antibody, and betweenantibody and antigen), then an amino acid or peptide insertion may beaccomplished by adding a peptide segment between residues x and y,leaving residues x and y unchanged. Alternatively, either or bothresidues x and y may be deleted and replaced by a peptide segment of >2residues.

Often, residues x and y, and/or intervening residues in the parentantibody, may be involved in significant intramolecular andintermolecular contacts. In this case, these interactions may bemaintained or replaced with residues contributing similar interactions,while allowing for an inserted residue or peptide to exit and reenterthe chain. This may be accomplished by substituting the two residues xand y and/or intervening residues in the parent antibody with randomresidues, which can be subsequently subjected to affinity screening (orscreening for other biological activities) to identify variants withimproved affinity.

This systematic method is illustrated in FIG. 3 for example, whereresidues D41 and E42 in the VEGF antigen were identified as potentialcandidates for interacting with introduced residues in CDR H3 of theheavy chain variable domain of the parent antibody.

Thus, as illustrated in FIGS. 4 and 5, D41 of the VEGF antigen is ableto form an ion pair with inserted residue R104c in CDR H3 of variantantibody Y0313-2 of the Example below. FIG. 5 further shows how residueV104 in variant antibody Y0313-2 is able to form a hydrophobicinteraction with residues 93 to 95 of the VEGF antigen. Thus, it can beseen that one identifies potential areas where the contacts betweenantigen and antibody can be improved, so as to increase the affinity ofthe antibody variant.

Generally one makes changes in hypervariable regions proximal to antigenwhen the antigen and antibody are complexed together. For example, thehypervariable region of the parent antibody which may be modified asdisclosed herein generally has one or more amino acid residues withinabout 20 Å of one or more amino acid residues of the antigen. Thehypervariable region to be altered herein may be one which, in theparent antibody, does not make significant contact with antigen (i.e. anon-contacting hypervariable region can be modified to become acontacting hypervariable region). Preferably however, the hypervariableregion to be modified does contact antigen and the method herein servesto increase the contacts between the antigen and the already-contactinghypervariable region.

In another embodiment, one may identify hypervariable region residueswhich interact with antigen by alanine-scanning mutagenesis of theantigen and/or parent antibody (Muller et al. Structure 6(9): 1153-1167(1998)) or by other means. Hypervariable regions identified ascontacting antigen are candidates for amino acid insertion(s) as hereindisclosed.

Nucleic acid molecules encoding amino acid sequence variants areprepared by a variety of methods known in the art. These methodsinclude, but are not limited to, oligonucleotide-mediated (orsite-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis ofan earlier prepared variant or a non-variant version of the parentantibody. The preferred method for making variants is site directedmutagenesis (see, e.g., Kunkel, Proc. Natl. Acad. Sci. USA 82:488(1985)). Moreover, a nucleic acid sequence can be made synthetically,once the desired amino acid sequence is arrived at conceptually. One canalso make the antibody variant by peptide synthesis, peptide ligation orother methods.

Following production of the antibody variant, the activity of thatmolecule relative to the parent antibody may be determined. As notedabove, this may involve determining the binding affinity and/or otherbiological activities of the antibody. In a preferred embodiment of theinvention, a panel of antibody variants are prepared and are screenedfor binding affinity for the antigen and/or potency in one or morebiological activity assays. One or more of the antibody variantsselected from an initial screen are optionally subjected to one or morefurther biological activity assays to confirm that the antibodyvariant(s) have improved activity in more than one assay.

One preferred method of making and screening insertion mutants involvesdisplaying antibody variants on the surface of filamentous bacteriophageand selecting antibody variants based on their affinity for antigen, bytheir kinetics of dissociation (off-rate) from antigen, or some otherscreen for antibody affinity or potency. This was the method used toidentify antibody variants with enhanced biological activity in theExample below.

Aside from the above insertions in the hypervariable region of theparent antibody one may make other alterations in the amino acidsequences of one or more of the hypervariable regions. For example, theabove amino acid insertions may be combined with deletions orsubstitutions of other hypervariable region residues. Moreover, one ormore alterations (e.g. substitutions) of FR residues may be introducedin the parent antibody where these result in an improvement in thebinding affinity of the antibody variant for the antigen. Examples offramework region residues to modify include those which non-covalentlybind antigen directly (Amit et al. Science 233:747-753 (1986)); interactwith/effect the conformation of a CDR (Chothia et al. J. Mol. Biol.196:901-917 (1987)); and/or participate in the V_(L)-V_(H) interface (EP239 400B1). Such amino acid sequence alterations may be present in theparent antibody, may be made simulateously with the amino acidinsertion(s) herein or may be made after a variant with an amino acidinsertion is generated.

The antibody variants may be subjected to other modifications,oftentimes depending on the intended use of the antibody. Suchmodifications may involve further alteration of the amino acid sequence,fusion to heterologous polypeptide(s) and/or covalent modification. Withrespect to amino acid sequence alterations, exemplary modifications areelaborated above. For example, any cysteine residue not involved inmaintaining the proper conformation of the antibody variant also may besubstituted, generally with serine, to improve the oxidative stabilityof the molecule and prevent aberrant cross linking. Conversely, cysteinebond(s) may be added to the antibody to improve its stability(particularly where the antibody is an antibody fragment such as an Fvfragment). Another type of amino acid variant has an alteredglycosylation pattern. This may be achieved by deleting one or morecarbohydrate moieties found in the antibody, and/or adding one or moreglycosylation sites that are not present in the antibody. Glycosylationof antibodies is typically either N-linked or O-linked. N-linked refersto the attachment of the carbohydrate moiety to the side chain of anasparagine residue. The tripeptide sequences asparagine-X-serine andasparagine-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of the carbohydratemoiety to the asparagine side chain. Thus, the presence of either ofthese tripeptide sequences in a polypeptide creates a potentialglycosylation site. O-linked glycosylation refers to the attachment ofone of the sugars N-aceylgalactosamine, galactose, or xylose to ahydroxyamino acid, most commonly serine or threonine, although5-hydroxyproline or 5-hydroxylysine may also be used. Addition ofglycosylation sites to the antibody is conveniently accomplished byaltering the amino acid sequence such that it contains one or more ofthe above-described tripeptide sequences (for N-linked glycosylationsites). The alteration may also be made by the addition of, orsubstitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Techniques for producing antibodies, which may be the parent antibodyand therefore require modification according to the techniqueselaborated herein, follow:

A. Antibody Preparation

(i) Antigen Preparation

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g. theextracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule. Otherantigens and forms thereof useful for preparing antibodies will beapparent to those in the art.

(ii) Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(iii) Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. Recombinant production of antibodies will be described in moredetail below.

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

(iv) Humanized and Human Antibodies

A humanized antibody has one or more amino acid residues introduced intoit from a source which is non-human. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman FR for the humanized antibody (Sims et al., J. Immunol., 151:2296(1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another methoduses a particular FR derived from the consensus sequence of all humanantibodies of a particular subgroup of light or heavy chains. The sameFR may be used for several different humanized antibodies (Carter etal., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J.Immnol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993); and Duchosal et al. Nature 355:258(1992). Human antibodies can also be derived from phage-displaylibraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks etal., J. Mol. Biol., 222:581-597 (1991); Vaughan et al. Nature Biotech14:309 (1996)).

(v) Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992) and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185.

(vi) Multispecific Antibodies

Multispecific antibodies have binding specificities for at least twodifferent antigens. While such molecules normally will only bind twoantigens (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by thisexpression when used herein. Examples of BsAbs include those with onearm directed against a tumor cell antigen and the other arm directedagainst a cytotoxic trigger molecule such as anti-FcγRI/anti-CD15,anti-p185^(HER2)/FcγRIII (CD16), anti-CD3/anti-malignant B-cell (1D10),anti-CD3/anti-p185^(HER2), anti-CD3/anti-p97, anti-CD3/anti-renal cellcarcinoma, anti-CD3/anti-OVCAR-3, anti-CD3/L-D1 (anti-colon carcinoma),anti-CD3/anti-melanocyte stimulating hormone analog, anti-EGFreceptor/anti-CD3, anti-CD3/anti-CAMA1, anti-CD3/anti-CD19,anti-CD3/MoV18, anti-neural cell ahesion molecule (NCAM)/anti-CD3,anti-folate binding protein (FBP)/anti-CD3, anti-pan carcinomaassociated antigen (AMOC-31)/anti-CD3; BsAbs with one arm which bindsspecifically to a tumor antigen and one arm which binds to a toxin suchas anti-saporin/anti-Id-1, anti-CD22/anti-saporin,anti-CD7/anti-saporin, anti-CD38/anti-saporin, anti-CEA/anti-ricin Achain, anti-CEA/anti-vinca alkaloid; BsAbs for converting enzymeactivated prodrugs such as anti-CD30/anti-alkaline phosphatase (whichcatalyzes conversion of mitomycin phosphate prodrug to mitomycinalcohol); BsAbs which can be used as fibrinolytic agents such asanti-fibrin/anti-tissue plasminogen activator (tPA),anti-fibrin/anti-urokinase-type plasminogen activator (uPA); BsAbs fortargeting immune complexes to cell surface receptors such as anti-lowdensity lipoprotein (LDL)/anti-Fc receptor (e.g. FcγRI, FcγRII orFcγRIII); BsAbs for use in therapy of infectious diseases such asanti-CD3/anti-herpes simplex virus (HSV), anti-T-cell receptor:CD3complex/anti-influenza, anti-FcγR/anti-HIV; BsAbs for tumor detection invitro or in vivo such as anti-CEA/anti-EOTUBE, anti-CEA/anti-DPTA,anti-p185^(HER2)/anti-hapten; BsAbs as vaccine adjuvants; and BsAbs asdiagnostic tools such as anti-rabbit IgG/anti-ferritin, anti-horseradish peroxidase (HRP)/anti-hormone, anti-somatostatin/anti-substanceP, anti-HRP/anti-FITC. Examples of trispecific antibodies includeanti-CD3/anti-CD4/anti-CD37, anti-CD3/anti-CD5/anti-CD37 andanti-CD3/anti-CD8/anti-CD37. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the C_(H)3domain of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

(vii) Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance the effectiveness of the antibodyin treating cancer, for example. For example cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992)and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodieswith enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch 53:2560-2565 (1993). Alternatively, an antibody can beengineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al. Anti-CancerDrug Design 3:219-230 (1989).

(viii) Immunoconjugates

The invention also pertains to immunoconjugates comprising the antibodydescribed herein conjugated to a cytotoxic agent such as achemotherapeutic agent, toxin (e.g. an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof which can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugate antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y and¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g. avidin) which is conjugatedto a cytotoxic agent (e.g. a radionucleotide).

(ix) Immunoliposomes

The antibody variants disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al. J. Biol. Chem.257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al. J. National Cancer Inst.81(19)1484 (1989)

(x) Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

The antibody of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g. a peptidyl chemotherapeutic agent, see WO81/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as beta-galactosidase andneuraminidase useful for converting glycosylated prodrugs into freedrugs; beta-lactamase useful for converting drugs derivatized withbeta-lactams into free drugs; and penicillin amidases, such aspenicillin V amidase or penicillin G amidase, useful for convertingdrugs derivatized at their amine nitrogens with phenoxyacetyl orphenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”,can be used to convert the prodrugs of the invention into free activedrugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzymeconjugates can be prepared as described herein for delivery of theabzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the antibodyvariant by techniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature, 312: 604-608 (1984)).

(xi) Antibody-Salvage Receptor Binding Epitope Fusions.

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody, to increase tumorpenetration, for example. In this case, it may be desirable to modifythe antibody fragment in order to increase its serum half life. This maybe achieved, for example, by incorporation of a salvage receptor bindingepitope into the antibody fragment (e.g. by mutation of the appropriateregion in the antibody fragment or by incorporating the epitope into apeptide tag that is then fused to the antibody fragment at either end orin the middle, e.g., by DNA or peptide synthesis).

The salvage receptor binding epitope preferably constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to an analogous position of the antibodyfragment. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or V_(H) region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the C_(L) region or V_(L)region, or both, of the antibody fragment. See, e.g., U.S. Pat. No.5,739,277, issued Apr. 14, 1998.

(xii) Covalent Modifications

Covalent modifications of the antibody are included within the scope ofthis invention. They may be made by chemical synthesis or by enzymaticor chemical cleavage of the antibody, if applicable. Other types ofcovalent modifications of the antibody are introduced into the moleculeby reacting targeted amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

Removal of any carbohydrate moieties present on the antibody may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described by Hakimuddin, etal. Arch. Biochem. Biophys. 259:52 (1987) and by Edge et al. Anal.Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties onantibodies can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. Meth. Enzymol. 138:350(1987).

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

B. Vectors, Host Cells and Recombinant Methods

The invention also provides isolated nucleic acid encoding an antibodyvariant as disclosed herein, vectors and host cells comprising thenucleic acid, and recombinant techniques for the production of theantibody variant.

For recombinant production of the antibody variant, the nucleic acidencoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the monoclonal antibody variant is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the antibody variant). Many vectors areavailable. The vector components generally include, but are not limitedto, one or more of the following: a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence.

(i) Signal Sequence Component

The antibody variant of this invention may be produced recombinantly notonly directly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the native antibody signal sequence, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, lpp,or heat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,α factor leader (including Saccharomyces and Kluyveromyces α-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or the signal described in WO 90/13646. In mammalian cellexpression, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody variant.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding antibody, wild-type DHFR protein, and another selectable markersuch as aminoglycoside 3′-phosphotransferase (APH) can be selected bycell growth in medium containing a selection agent for the selectablemarker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin,or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodynucleic acid. Promoters suitable for use with prokaryotic hosts includethe phoA promoter, beta-lactamase and lactose promoter systems, alkalinephosphatase, a tryptophan (trp) promoter system, and hybrid promoterssuch as the tac promoter. However, other known bacterial promoters aresuitable. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theantibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phospho-fructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. Alternatively, the rous sarcoma virus long terminal repeatcan be used as the promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding the antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, alpha-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elementsfor activation of eukaryotic promoters. The enhancer may be spliced intothe vector at a position 5′ or 3′ to the antibody-encoding sequence, butis preferably located at a site 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibody arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce the antibody variant of this inventionmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM),Sigma) are suitable for culturing the host cells. In addition, any ofthe media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes etal., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866;4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S.patent Re. 30,985 may be used as culture media for the host cells. Anyof these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

(ix) Antibody Purification

When using recombinant techniques, the antibody variant can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody variant is produced intracellularly, as a firststep, the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody variantis secreted into the medium, supernatants from such expression systemsare generally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody variant.Protein A can be used to purify antibodies that are based on human γ1,γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13(1983)). Protein G is recommended for all mouse isotypes and for humanγ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which theaffinity ligand is attached is most often agarose, but other matricesare available. Mechanically stable matrices such as controlled poreglass or poly(styrenedivinyl)benzene allow for faster flow rates andshorter processing times than can be achieved with agarose. Where theantibody variant comprises a C_(H)3 domain, the Bakerbond ABX™ resin (J.T. Baker, Phillipsburg, N.J.) is useful for purification. Othertechniques for protein purification such as fractionation on anion-exchange column, ethanol precipitation, Reverse Phase HPLC,chromatography on silica, chromatography on heparin SEPHAROSE™chromatography on an anion or cation exchange resin (such as apolyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation are also available depending on the antibodyvariant to be recovered.

C. Pharmaceutical Formulations

Therapeutic formulations of the antibody variant are prepared forstorage by mixing the antibody variant having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide an immunosuppressiveagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody variant, which matrices arein the form of shaped articles, e.g., films, or microcapsule. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

D. Non-Therapeutic Uses for the Antibody Variant

The antibody variants of the invention may be used as affinitypurification agents. In this process, the antibodies are immobilized ona solid phase such a Sephadex resin or filter paper, using methods wellknown in the art. The immobilized antibody variant is contacted with asample containing the antigen to be purified, and thereafter the supportis washed with a suitable solvent that will remove substantially all thematerial in the sample except the antigen to be purified, which is boundto the immobilized antibody variant. Finally, the support is washed withanother suitable solvent, such as glycine buffer, pH 5.0, that willrelease the antigen from the antibody variant.

The variant antibodies may also be useful in diagnostic assays, e.g.,for detecting expression of an antigen of interest in specific cells,tissues, or serum.

For diagnostic applications, the antibody variant typically will belabeled with a detectable moiety. Numerous labels are available whichcan be generally grouped into the following categories:

(a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The antibodyvariant can be labeled with the radioisotope using the techniquesdescribed in Current Protocols in Immunology, Volumes 1 and 2, Coligenet al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991) for exampleand radioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the antibody variant using thetechniques disclosed in Current Protocols in Immunology, supra, forexample. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. VanVunakis), Academic press, New York, 73:147-166 (1981).

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate aschromogenic substrate; and

(iii) beta-D-galactosidase (beta-D-Gal) with a chromogenic substrate(e.g., p-nitrophenyl-beta-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-beta-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

Sometimes, the label is indirectly conjugated with the antibody variant.The skilled artisan will be aware of various techniques for achievingthis. For example, the antibody variant can be conjugated with biotinand any of the three broad categories of labels mentioned above can beconjugated with avidin, or vice versa. Biotin binds selectively toavidin and thus, the label can be conjugated with the antibody variantin this indirect manner. Alternatively, to achieve indirect conjugationof the label with the antibody variant, the antibody variant isconjugated with a small hapten (e.g., digoxin) and one of the differenttypes of labels mentioned above is conjugated with an anti-haptenantibody variant (e.g., anti-digoxin antibody). Thus, indirectconjugation of the label with the antibody variant can be achieved.

In another embodiment of the invention, the antibody variant need not belabeled, and the presence thereof can be detected using a labeledantibody which binds to the antibody variant.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyze for binding with a limited amountof antibody variant. The amount of antigen in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyze that are boundto the antibodies may conveniently be separated from the standard andanalyze which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyze is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyze, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

For immunohistochemistry, the tumor sample may be fresh or frozen or maybe embedded in paraffin and fixed with a preservative such as formalin,for example.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody variant is labeled with a radionuclide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography.

E. Diagnostic Kits

As a matter of convenience, the antibody variant of the presentinvention can be provided in a kit, i.e., a packaged combination ofreagents in predetermined amounts with instructions for performing thediagnostic assay. Where the antibody variant is labeled with an enzyme,the kit will include substrates and cofactors required by the enzyme(e.g., a substrate precursor which provides the detectable chromophoreor fluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g., a block buffer or lysis buffer) and thelike. The relative amounts of the various reagents may be varied widelyto provide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients which on dissolution will provide a reagent solution havingthe appropriate concentration.

F. In Vivo Uses for the Antibody Variant

For therapeutic applications, the antibody variants of the invention areadministered to a mammal, preferably a human, in a pharmaceuticallyacceptable dosage form such as those discussed above, including thosethat may be administered to a human intravenously as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes. Theantibodies also are suitably administered by intra-tumoral,peri-tumoral, intra-lesional, or peri-lesional routes, to exert local aswell as systemic therapeutic effects. The intra-peritoneal route isexpected to be particularly useful, for example, in the treatment ofovarian tumors. In addition, the antibody variant is suitablyadministered by pulse infusion, particularly with declining doses of theantibody variant. Preferably the dosing is given by injections, mostpreferably intravenous or subcutaneous injections, depending in part onwhether the administration is brief or chronic.

For the prevention or treatment of disease, the appropriate dosage ofantibody variant will depend on the type of disease to be treated, theseverity and course of the disease, whether the antibody variant isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody variant, andthe discretion of the attending physician. The antibody variant issuitably administered to the patient at one time or over a series oftreatments.

The example herein concerns an anti-VEGF antibody. Anti-VEGF antibodiesare useful in the treatment of various neoplastic and non-neoplasticdiseases and disorders. Neoplasms and related conditions that areamenable to treatment include breast carcinomas, lung carcinomas,gastric carcinomas, esophageal carcinomas, colorectal carcinomas, livercarcinomas, ovarian carcinomas, thecomas, arrhenoblastomas, cervicalcarcinomas, endometrial carcinoma, endometrial hyperplasia,endometriosis, fibrosarcomas, choriocarcinoma, head and neck cancer,nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi'ssarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma,hemangioblastoma, pancreas carcinomas, retinoblastoma, astrocytoma,glioblastoma, Schwannoma, oligodendroglioma, medulloblastoma,neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas,urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, renal cellcarcinoma, prostate carcinoma, abnormal vascular proliferationassociated with phakomatoses, edema (such as that associated with braintumors), and Meigs' syndrome.

Non-neoplastic conditions that are amenable to treatment includerheumatoid arthritis, psoriasis, atherosclerosis, diabetic and otherproliferative retinopathies including retinopathy of prematurity,retrolental fibroplasia, neovascular glaucoma, age-related maculardegeneration, thyroid hyperplasias (including Grave's disease), cornealand other tissue transplantation, chronic inflammation, lunginflammation, nephrotic syndrome, preeclampsia, ascites, pericardialeffusion (such as that associated with pericarditis), and pleuraleffusion.

Age-related macular degeneration (AMD) is a leading cause of severevisual loss in the elderly population. The exudative form of AMD ischaracterized by choroidal neovascularization and retinal pigmentepithelial cell detachment. Because choroidal neovascularization isassociated with a dramatic worsening in prognosis, the VEGF antibodiesof the present invention are expected to be especially useful inreducing the severity of AMD.

Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g., 0.1-20 mg/kg) of antibody variant is an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. A typicaldaily dosage might range from about 1 μg/kg to 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays.

The antibody variant composition will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the antibody variant to beadministered will be governed by such considerations, and is the minimumamount necessary to prevent, ameliorate, or treat a disease or disorder.The antibody variant need not be, but is optionally formulated with oneor more agents currently used to prevent or treat the disorder inquestion. The effective amount of such other agents depends on theamount of antibody variant present in the formulation, the type ofdisorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asused hereinbefore or about from 1 to 99% of the heretofore employeddosages.

G. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for treating the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The active agent in the composition is the antibody variant.The label on, or associated with, the container indicates that thecomposition is used for treating the condition of choice. The article ofmanufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

Example 1

In this example, antibody variants containing randomized peptide insertswithin the antibody CDRs are prepared by phage display whichsubstantially improve the affinity of a humanized Fab for VEGF.Crystallography suggests that these changes result in an increasedcontact area with antigen.

VEGF:Fab X-ray Co-Crystal Structure:

A crystal structure of the complex between the VEGF antigen andanti-VEGF parent antibody was prepared as described in Muller et al.,Structure 6(9):1153-1167 (1998). The conclusion that the three VH CDRsare the main determinants of Fab binding to VEGF is supported by thehigh-resolution crystal structure of the VEGF:Fab (v36) complex. Inaddition, the major energetic determinants largely coincide with theprincipal contacting residues of the Fab in the complex.

Several randomized libraries were designed with a peptide insertionplaced in the antigen-contacting CDRs which, from the crystal structure,were expected to increase the potential contact between the antibody andthe antigen.

Design of CDR Random Loop-Insertion Libraries:

Based upon inspection of the VEGF:Fab crystal structure, it waspostulated that additional contacts, contributing additional bindingenergy between the Fab and VEGF, could be generated through the additionof peptide inserts within one or more CDRs of the Fab. Because thenature and relative contributions of such additional interactions wouldbe difficult to predict, randomized loop sequences (Xn) were directlyinserted into each of the four CDRs proximal to the existing VEGFbinding site using NNS codons, and a frameshifted Fab vector astemplate. The length of loop was chosen based upon distances in thecrystal structure between exit/entry points of the loop on thehypervariable region and possible interaction sites on the surface ofVEGF. In addition, one or more residues within each loop were deleted insome of these templates, as judged necessary to accommodate the newpeptide loop.

Three such loops were designed for VH1, including insertions of 4, 5, or6 residues between Y27 and T28. In VH2, two inserted peptides of 3 or 4residues were placed between Y54 and T55. Also in VH2, a 6-residuerandom peptide was used to replace residues T55 and H56. In VH3, a4-residue or 5-residue peptide was used to replace G104, and a 5-residueor 6-residue peptide was used to replace residues G104 and 5105.Finally, in VL3, a random peptide of either 4 or 6 residues was insertedbetween S92 and T93.

Second-Generation Selections of Anti-VEGF Libraries:

Templates for random mutagenesis were constructed starting from theFab-g3 phagemid pY0192 (WO98/45331) and frameshift oligonucleotides(which prevent expression of a functional template Fab): YC-82, YC-85,YC-89, YC-92, YC-94, and YC-97 (Table 1).

TABLE 1 Frameshift oligos for CDR-insert template mutagenesis Oligo #Region Sequence SEQ. ID NO: YC-82 VL3C TGT CAA CAG TAT AGC T ACC GTG CCG TGG ACG SEQ. ID NO: 1 YC-85 VH1GCA GCT TCT GGC TAT G ACC TTC ACC AAC TAT G SEQ. ID NO: 2 YC-89 VH2GA TGG ATT AAC ACC TAT G ACC GGT GAA CCG ACC SEQ. ID NO: 3 YC-92 VH2GA TGG ATT AAC ACC TAT T GAA CCG ACC TAT GCT SEQ. ID G NO: 4 YC-94 VH3G TAC CCG CAC TAT TAT G AGC AGC CAC TGG TAT SEQ. ID TTC NO: 5 YC-97 VH3G TAC CCG CAC TAT TAT G AGC CAC TGG TAT TTC SEQ. ID NO: 6

The corresponding randomization oligonucleotides (which employ NNS atthe sites targeted for randomization) were YC-83, YC-84 in VL3; YC-86,YC-87, YC-88 in VH1; YC-90, YC91 and YC-93 in VH2; and YC-95, YC-96,YC-98, YC-99 in VH3. See Table 2 below.

TABLE 2 Random oligos for CDR-insert library constructions Oligo #Region (Comments) Sequence SEQ ID NO: YC-83 VL3 (insert 4C TGT CAA CAG TAT AGC NNS NNS NNS SEQ. ID residues)NNS ACC GTG CCG TGG ACG NO: 7 YC-84 VL3 (insert 6C TGT CAA CAG TAT AGC NNS NNS NNS SEQ. ID residues)NNS NNS NNS ACC GTG CCG TGG ACG NO: 8 YC-86 VH1 (insert 4GCA GCT TCT GGC TAT NNS NNS NNS SEQ. ID residues)NNS ACC TTC ACC AAC TAT G NO: 9 YC-87 VH1 (insert 5GCA GCT TCT GGC TAT NNS NNS NNS SEQ. ID residues)NNS NNS ACC TTC ACC AAC TAT G NO: 10 YC-88 VH1 (insert 6GCA GCT TCT GGC TAT NNS NNS NNS SEQ. ID residues)NNS NNS NNS ACC TTC ACC AAC TAT G NO: 11 YC-90 VH2 (insert 3GA TGG ATT AAC ACC TAT NNS NNS NNS SEQ. ID residues) ACC GGT GAA CCG ACCNO: 12 YC-91 VH2 (insert 4 GA TGG ATT AAC ACC TAT NNS NNS NNS SEQ. IDresidues) NNS ACC GGT GAA CCG ACC NO: 13 YC-93 VH2 (insert 6GA TGG ATT AAC ACC TAT NNS NNS NNS SEQ. ID residues)NNS NNS NNS GAA CCG ACC TAT GCT G NO: 14 YC-95 VH3 (insert 4G TAC CCG CAC TAT TAT NNS NNS NNS SEQ. ID residues)NNS AGC AGC CAC TGG TAT TTC NO: 15 YC-96 VH3 (insert 5G TAC CCG CAC TAT TAT NNS NNS NNS SEQ. ID residues)NNS NNS AGC AGC CAC TGG TAT TTC NO: 16 YC-98 VH3 (insert 5G TAC CCG CAC TAT TAT NNS NNS NNS SEQ. ID residues)NNS NNS AGC CAC TGG TAT TTC NO: 17 YC-99 VH3 (insert 6G TAC CCG CAC TAT TAT NNS NNS NNS SEQ. ID residues)NNS NNS NNS AGC CAC TGG TAT TTC NO: 18

The resulting transformants yielded libraries with complexities rangingfrom 6×10⁷ to 5×10⁸ suggesting that the libraries were comprehensive incovering all possible variants.

Each library was sorted separately for the first round; thereafter,libraries with the same site of insertion were combined and sortedtogether as one. Therefore, library YC-83 was combined with libraryYC-84; library YC-86 with libraries YC-87 and YC-88; library YC-90 withYC-91; library YC-95 with YC-96; and library YC-98 with YC-99. Theselibraries were sorted essentially as described in WO98/45331, except theincubation with PBS/TWEEN 20® buffer after phage binding was carried outas described in Table 3.

TABLE 3 Conditions for secondary selections of Fab variants round ofincubation time incubation selection (hr) incubation solution temp.(°C.) 1 0 0 room temp. 2 1 ELISA buffer room temp. 3 2 1 μM VEGF/ELISAroom temp. 4 18 1 μM VEGF/ELISA room temp. 5 37 1 μM VEGF/ELISA roomtemp. 6 17 hr@R.T./30 h@ same as above room temp./ 37° C. 37° C. 7 63same as above 37° C. 8 121 same as above 37° C.

ELISA buffer contained 0.5% bovine serum albumin and 0.05% TWEEN 20® inPBS. VEGF was included in the incubation buffer to minimize rebinding ofphage to VEGF coated on the surface of the plate.

Sorting of some of these libraries yielded VEGF-binding phageenrichments over 5 to 8 rounds of selection. After five to eight roundsof selections, ten to twenty clones from each library were isolated fromcarbenicillin containing plates harboring E. coli (XL1) colonies whichhad been infected with an eluted phage pool. Colonies were isolated andgrown with helper phage to obtain single-stranded DNA for sequencing.Clones were picked from those libraries that enriched for DNAsequencing. The results are shown in Table 4. Libraries showing noenrichment were not sequenced.

TABLE 4 Summary of CDR Insertion Libraries No. of added Oligos Site ofresidues Stop oligo Insert oligo CDR Insertion Net Total YC-85 YC-86 H1Y27{circumflex over ( )}T28 4 4 YC-85 YC-87 H1 Y27{circumflex over( )}T28 5 5 YC-85 YC-88 H1 Y27{circumflex over ( )}T28 6 6 YC-89 YC-90H2 Y54{circumflex over ( )}T55 3 3 YC-89 YC-91 H2 Y54{circumflex over( )}T55 4 4 YC-92 YC-93 H2 Y54{circumflex over ( )}E57 4 6 YC-94 YC-95H3 Y103{circumflex over ( )}S105 3 4 YC-94 YC-96 H3 Y103{circumflex over( )}S105 4 5 YC-97 YC-98 H3 Y103{circumflex over ( )}S106 3 5 YC-97YC-99 H3 Y103{circumflex over ( )}S106 4 6 YC-82 YC-83 L3 S92{circumflexover ( )}T93 4 4 YC-82 YC-84 L3 S92{circumflex over ( )}T93 6 6

For VH1, only library YC-86 showed enrichment. Sequencing revealed that,although a 4-residue insert was designed in this library, all of thesequenced clones contained no net insertion, but instead point mutationsat T28 and F29. This suggests that this antibody is relativelyintolerant of insertions in this hypervariable region.

A similar result was seen for the VH2 libraries, where only libraryYC-90 showed enrichment. Again, clones found were either wild-type(Y0192) or a point mutant, Y54W. This suggests that this antibody isalso relatively intolerant of insertions in the VH2 CDR.

Again, a similar result was obtained for the VL3 libraries. In thiscase, only library YC-83 showed enrichment, and the selected clones hadpoint mutations at T93 and/or V94, rather than the designed insertion.This suggests that this antibody is also relatively intolerant ofinsertions in the VL3 CDR.

In contrast, two VH3 libraries showed enrichment: YC-95 and YC-98.Moreover, sequencing of selected clones showed that the Fab variantsindeed contained insertion sequences.

Amino acid sequences of anti-VEGF variants from the various librariesare shown in Tables 5-15 below. The sequence of the randomized regiononly is shown as deduced from DNA sequencing. Sites where randomizedinserted sequences were made are shown in bold. An asterisk denotes acontaminating phagemid from another library.

TABLE 5 Protein sequences of anti-VEGF variants from library YC-86Round 7 (VEGF eluted phage) VH1 sequence Name (residues 26-35)SEQ ID NO: (# clones/10) Y0241-1 GYDFTNYGIN SEQ. ID NO: 19 4 Y0241-6GYDYTNYGIN SEQ. ID NO: 20 3 Y0241-7 GYDWTNYGIN SEQ. ID NO: 21 3

TABLE 6 Protein sequences of anti-VEGF variants from library YC-90Round 7 (VEGF eluted phage) VH2 sequence Name (residues 50-62)SEQ ID NO: (# clones/10) Y0242-1 WINTWTGEPTYAA SEQ. ID NO: 22 4 *Y0192 6

TABLE 7 Protein sequences of anti-VEGF variants from library YC-83Round 7 (VEGF eluted phage) VL3 sequence Name (residues 89-97)SEQ ID NO: (# clones/9) Y0241-2 QQYSATPWT SEQ. ID NO: 23 1 Y0241-3QQYSNVPWT SEQ. ID NO: 24 3 Y0241-4 QQYSAVPWT SEQ. ID NO: 25 4 Y0241-5QQYSSVPWT SEQ. ID NO: 26 1

TABLE 8 Protein sequences of anti-VEGF variants from library YC-95Round 5 (VEGF eluted phage) VH3 sequence (residues Name 99-111) +insertions SEQ ID NO: (# clones/10) Y0228-1 YPHYYAKERSSHWYFDVSEQ. ID NO: 27 1 Y0228-2 YPHYYVGETSSHWYFDV SEQ. ID NO: 28 1 Y0228-3YPHYYARDRSSHWYFDV SEQ. ID NO: 29 1 Y0228-4 YPHYYERDGKSSHWYFDVSEQ. ID NO: 30 1 Y0228-5 YPHYYRNEKSSHWYFDV SEQ. ID NO: 31 1 Y0228-6YPHYYVGEQSSHWYFDV SEQ. ID NO: 32 1 Y0228-7 YPHYYQRDRSSHWYFDVSEQ. ID NO: 33 1 Y0228-8 YPHYYQKQSKSSHWYFDV SEQ. ID NO: 34 1 Y0228-9YPHYYQNEGPSSHWYFDV SEQ. ID NO: 35 1 Y0228-10 YPHYYGNHRSSHWYFDVSEQ. ID NO: 36 1

TABLE 9 Protein sequences of anti-VEGF variants from library YC-95Round 5 (HCl eluted phage) VH3 sequence (residues Name 99-111) +insertions SEQ ID NO: (# clones/10) Y0229-1 YPHYYRTEKSSHWYFDVSEQ. ID NO: 37 1 Y0229-2 YPHYYLKDRSSHWYFDV SEQ. ID NO: 38 1 Y0229-4YPHYYQDEKSSHWYFDV SEQ. ID NO: 39 1 Y0229-5 YPHYYVGEKSSHWYFDVSEQ. ID NO: 40 1 Y0229-6 YPHYYRDERSSHWYFDV SEQ. ID NO: 41 1 Y0229-7YPHYYTYDKSSHWYFDV SEQ. ID NO: 42 1 Y0229-8 YPHYYHTRGGSSHWYFDVSEQ. ID NO: 43 1 Y0229-9 YPHYYLNDKSSHWYFDV SEQ. ID NO: 44 1 Y0229-10YPHYYYRDRSSHWYFDV SEQ. ID NO: 45 1 *Y0239-1 1

TABLE 10 Protein sequences of anti-VEGF variants from library YC-95Round 7 (HCl eluted phage) VH3 sequence (residues Name 99-111) +insertions SEQ ID NO: (# clones/10) Y0239-1 YPHYYRNERSSHWYFDVSEQ. ID NO: 46 1 Y0239-2 YPHYYKNDKSSHWYFDV SEQ. ID NO: 47 1 Y0239-3YPHYYLADRSSHWYFDV SEQ. ID NO: 48 1 Y0239-4 YPHYYVNERSSHWYFDVSEQ. ID NO: 49 1 Y0239-5 YPHYYLKDKSSHWYFDV SEQ. ID NO: 50 1 Y0239-6YPHYYLKDGRSSHWYFDV SEQ. ID NO: 51 1 Y0239-7 YPHYYERDGRSSHWYFDVSEQ. ID NO: 52 1 Y0239-8 YPHYYLRDGRSSHWYFDV SEQ. ID NO: 53 1 Y0239-9YPHYYLGESSHWYFDV SEQ. ID NO: 54 1 Y0239-10 YPHYYLGEKSSHWYFDVSEQ. ID NO: 55 1

TABLE 11 Protein sequences of anti-VEGF variants from library YC-95Round 8 (HCl eluted phage) VH3 sequence (residues Name 99-111) +insertions SEQ ID NO: (# clones/10) Y0261-1 YPHYYLKDRRSSHWYFDVSEQ. ID NO: 56 2 Y0261-2 YPHYYLKDGMSSHWYFDV SEQ. ID NO: 57 2 *Y0239-4 1*Y0239-9 5

TABLE 12 Protein sequences of anti-VEGF variants from library YC-98Round 5 (VEGF eluted phage) VH3 sequence (residues Name 99-111) +insertions SEQ ID NO: (# clones/10) Y0228-11 YPHYYEKQRKSHWYFDVSEQ. ID NO: 58 1 Y0228-12 YPHYYKEDKKSHWYFDV SEQ. ID NO: 59 1 Y0228-13YPHYYSHQKRSHWYEDV SEQ. ID NO: 60 1 Y0228-14 YPHYYSGERESHWYFDVSEQ. ID NO: 61 1 Y0228-15 YPHYYQSEGRSHWYFDV SEQ. ID NO: 62 1 Y0228-16YPHYYSVEGGSHWYFDV SEQ. ID NO: 63 1 Y0228-17 YPHYYPSPRGSHWYFDVSEQ. ID NO: 64 1 Y0228-18 YPHYYQRNGKSHWYFDV SEQ. ID NO: 65 1 Y0228-19YPHYYAREGGSHWYFDV SEQ. ID NO: 66 1 Y0228-20 YPHYYSNERKSHWYFDVSEQ. ID NO: 67 1

TABLE 13 Protein sequences of anti-VEGF variants from library YC-98Round 5 (HCl eluted phage) VH3 sequence (residues Name 99-111) +insertions SEQ ID NO: (# clones/10) Y0229-11 YPHYYRGDRKSHWYFDVSEQ. ID NO: 68 1 Y0229-12 YPHYYSDEKKSHWYFDV SEQ. ID NO: 69 1 Y0229-13YPHYYRSQRKSHWYFDV SEQ. ID NO: 70 1 Y0229-14 YPHYYAWRDRRSHWYFDVSEQ. ID NO: 71 1 Y0229-15 YPHYYANRERKSHWYFDV SEQ. ID NO: 72 1 Y0229-16YPHYYVNDKTSHWYFDV SEQ. ID NO: 73 1 Y0229-17 YPHYYVEETESHWYFDVSEQ. ID NO: 74 1 Y0229-18 YPHYYEKERKSHWYFDV SEQ. ID NO: 75 1 Y0229-19YPHYYSHERVSHWYFDV SEQ. ID NO: 76 1

TABLE 14 Protein sequences of anti-VEGF variants from library YC-98Round 7 (HCl eluted phage) VH3 sequence (residues Name 99-111) +insertions SEQ ID NO: (# clones/10) Y0239-11 YPHYYRDERESHWYFDVSEQ. ID NO: 77 1 Y0239-12 YPHYYAHEKKSHWYFDV SEQ. ID NO: 78 1 Y0239-13YPHYYLKDRKSHWYFDV SEQ. ID NO: 79 1 Y0239-14 YPHYYQHDRTSHWYFDVSEQ. ID NO: 80 1 Y0239-15 YPHYYVTDRKSHWYFDV SEQ. ID NO: 81 1 Y0239-16YPHYYLRDKKSHWYFDV SEQ. ID NO: 82 1 Y0239-17 YPHYYSHERKSHWYFDVSEQ. ID NO: 83 1 Y0239-18 YPHYYLNERKSHWYFDV SEQ. ID NO: 84 1 Y0239-19YPHYYVNERKSHWYFDV SEQ. ID NO: 85 2 Y0240-1 YPHYYLTDHKSHWYFDVSEQ. ID NO: 86 1

TABLE 15 Protein sequences of anti-VEGF variants from library YC-98Round 8 (HCl eluted phage) VH3 sequence (residues Name 99-111) +insertions SEQ ID NO: (# clones/10) Y0261-4 YPHYYLKDGKKSHWYFDVSEQ. ID NO: 87 1 Y0261-5 YPHYYRRDKKSHWYFDV SEQ. ID NO: 88 1 Y0261-6YPHYYLKDKKSHWYFDV SEQ. ID NO: 89 1 Y0261-7 YPHYYLHDRKSHWYFDVSEQ. ID NO: 90 1 Y0261-8 YPHYYLSDKKSHWYFDV SEQ. ID NO: 91 1 Y0239-19YPHYYVNERKSHWYFDV SEQ. ID NO: 92 1 *Y0239-13 1 *Y0239-16 3

In order to quantify relative antigen-binding affinities, severalanti-VEGF variants' DNA were transformed into E. coli strain 34B8,expressed as Fab, and purified by passing the periplasmic shockatethrough a protein G column (Pharmacia) as described in WO98/45331.

CDR combination Variant Y0313-2:

An attempt was made to improve antigen binding affinity by combining apreviously discovered CDR VH2 mutation with an insertion variantdescribed here. A mutagenic oligonucleotide, YC-107 (Table 16) was usedto combine insertion mutations found in CDR VH3, from clone Y0239-19,with VH2 CDR mutations T28D/N31H from clone Y0243-1 (WO98/45331) of CDRVH2.

TABLE 16 Mutagenesis oligo for adding a CDR insertion peptide(Table 16 discloses ″VNERK″ as SEQ ID NO: 100) Oligo # Region (Comments)Sequence SEQ. ID NO: YC-107 VH3 (insert VNERK from TAC CCG CAC TAT TATSEQ. ID NO: 93 library YC-98) GTG AAC GAG CGG AAG AGC CAC TGG TAT TTC

The resulting combined CDR variant was designated Y0313-2. A Fab proteinsample was prepared as described above for BIACORE™ analysis.

BIACORE™ Analysis:

The VEGF-binding affinities of Fab fragments were calculated fromassociation and dissociation rate constants measured using aBIACORE™-2000 surface plasmon resonance system (BIACORE™, Inc.,Piscataway, N.J.). A biosensor chip was activated for covalent couplingof VEGF using N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's (BIACORE™, Inc., Piscataway, N.J.) instructions. VEGF(8-109)was buffered exchanged into 20 mM sodium acetate, pH 4.8 and diluted toapproximately 50 μg/mL. Aliquots of VEGF were injected at a flow rate of2 μL/min to achieve approximately 700-1400 response units (RU) ofcoupled protein. A solution of 1 M ethanolamine was injected as ablocking agent.

For kinetics measurements, two-fold serial dilutions of Fab wereinjected in PBS/TWEEN buffer (0.05% TWEEN 20™ in phosphate bufferedsaline) at 25° C. at a flow rate of 10 μL/min. Equilibrium dissociationconstants, Kd's from SPR measurements were calculated as koff/kon (Table17).

TABLE 17 Kinetics of Fab-VEGF binding from BIACORE ™ measurements.Variant Kon(10⁴/M/s) koff (10⁻⁴/s) Kd(nM) Kd(wt)/Kd(mut) Y0192 4.1 1.212.9 -1-   Y0241-4 4.4 1.41 3.2 0.9 Y0241-7 4.6 1.28 3.0 1.0 Y0241-6 4.71.29 2.7 1.1 Y0242-1 4.7 0.86 1.8 1.6 Y0239-19 3.6 0.10 0.30 9.7 Y0239-83.8 0.18 0.50 5.8 Y0240-1 2.5 0.13 0.50 5.8 Y0239-2 3.6 1.64 4.6 0.6Y0239-12 5.7 0.34 0.6 4.8 Y0239-9 3.97 0.19 0.5 6.0 Y0261-6 4.4 0.25 0.65.0 Y0313-2 3.11 0.11 0.36 8.0

Results of SPR measurements demonstrated that affinity is mainlyenhanced through a slower dissociation rate (as opposed to fasterassociation).

For the insertion variant Y0239-19, an approximately 10-fold improvementin binding affinity was observed (Table 17). However, addition of theVH1 mutations did not further improve affinity, as indicated for thevariant Y0313-2.

Cell-Based Assay of VEGF:

Two Fab variants of the anti-VEGF antibody were tested for their abilityto antagonize VEGF (recombinant; version 1-165) in induction of thegrowth of HuVECs (human umbilical vein endothelial cells). The alamarblue assay (H. Gazzano-Santoro, et al. J Immunol Methods 202:163-171(1997)) was used to measure the metabolic activity of cells in responseto VEGF.

Two Fab variants of the anti-VEGF antibody were tested for their abilityto antagonize VEGF (recombinant; version 1-165) activity in induction ofthe growth of HuVECs (human umbilical vein endothelial cells). HuVECcells are seeded (1500/well) in a 96 well microtiter plate in completemedium (Cell Systems, Kirkland, Wash.) that has been coated with CellSystems attachment factor. The cells are allowed to attach for 24 hrs.On day 2, VEGF and Fab are diluted in assay medium(DMEM/F12+pennicillin/streptomycin, 0.1% gelatin). For the antibodyexperiments, a constant concentration of 5 ng/ml VEGF is added to allthe wells followed by the addition of various concentrations ofanti-VEGF Fab (approximately 10 μg/ml and dilutions). The VEGF and Fabincubate with the HUVEC cells for 2 days, after which 25 μl of alamarblue is added. Following a 4 hr incubation period, fluorescence is readon a Cytoflour Fluorescence Plate reader. The media used for theseassays is from Cell Systems.

The results (FIG. 2) show that the insertion variant Y0313-2 Fab hasroughly 100-fold enhanced potency over the original humanized antibody,F(ab)-12.

Crystallization and X-Ray Structure Determination of the Insert-FabY0313-2 in Complex with VEGF:

Crystals of VEGF in complex with the Fab fragment Y0313-2 were grown atroom temperature by vapor diffusion using the hanging drop method.Crystallization buffer containing 0.1 M sodium chloride, 20 mM Tris atpH 7.5, and the VEGF:Fab complex at a concentration of 8 mg/ml was mixedwith an equal amount of reservoir solution (15% PEG 4000, 5%isopropanol, 0.1M MES, pH 6.0, 0.2 M Citrate, 0.2 M Ammonium sulfate and1 mM SPADNS (2-(p-sulfophenylazo)-1,8-dihydroxy-3,6-naphthalenedisulphonic acid)). The resulting crystals belong to the monoclinicspace group P2 with cell parameters of a=107.6 Å, b=65.8 Å, c=123.8 Å,and β=93.4° and contain one VEGF-dimer bound to two Fab fragments in theasymmetric unit.

Prior to flash cooling with liquid nitrogen, crystals were dipped intoartificial mother liquor containing 20% glycerol. One diffraction dataset was collected from a single crystal at 100 K on a CCD detector atthe Advanced Light Source (Berkeley, Calif.). The data were processedusing MOSFLM (Leslie, A MOSFLM Users Guide, MRC-LMB, Cambridge (1994))and programs of the CCP4 suite (Collaborative Computing Project No. 4Acta Crystallog. sect. D, 50: 760-763 (1994)). The final data set was ofgood quality (Rsym=7.4%) with a completeness of 94.5% for allreflections between 25 Å and 2.8 Å resolution.

Initial phases for the complex were obtained by molecular replacement,using the constant domains and the variable domains of the Fab fragmentF(ab)-12 as separate search models. A model of the receptor bindingdomain of VEGF could be placed unambiguously in a resulting differencedensity map.

Refinement of the model with program X-PLOR (Bruenger et al. Science235: 458-460. (1987)) resulted in a final R-value of 21.2% with anR-free of 26.6% using all data between 2.8 Å and 25 Å.

New Antibody-Antigen Contacts in the Insert-Fab Complex with VEGF:

The results of x-ray crystallography show that the introduction of theinsert (Asn 104a, Glu 104b and Arg 104c (note: numbering of Y0313-2residues is sequential with inserted residues given a letter, ratherthan according to Kabat et al., supra) together with the twosubstitutions (G104V and S105K) enclosing it, increases the total amountof buried surface in the interface between VEGF and the antibody byabout 20% (see FIG. 4), as compared with the structure of the F(ab)-12complex (Muller et al., Structure 6(9):1153-1167 (1998)). The maincontributors for the enlargement of the contact area are residues Val104 and Arg 104c. Together, these two residues account for additional220 Å² of buried surface on the Fab fragment. The side chain of Val 104is packing tightly against the main chain of residues 93 to 95 of VEGF.The newly introduced Arg 104c forms a charged interaction with thecarboxyl group of Asp 41 of VEGF and is also in contact with the phenylring of Tyr 39 (see FIG. 5). Minor contributions to the interface aremade by the side chain of Lys 105 which is in the vicinity of the VEGFresidues Glu 44 and Tyr 45. The side chains of residues Asn 104a and Glu104b are pointing away from the interface and neither of themcontributes significantly to the interface between the Fab fragment andVEGF.

What is claimed is:
 1. An antibody variant of a parent antibody, whichantibody variant comprises an amino acid insertion in or adjacent to ahypervariable region of the parent antibody and has a binding affinityfor a target antigen which is at least about two fold stronger than thebinding affinity of the parent antibody for said antigen.
 2. Theantibody variant of claim 1 which has an amino acid insertion in ahypervariable region of the parent antibody.
 3. The antibody variant ofclaim 1 wherein the hypervariable region is Complementarity DeterminingRegion (CDR) H3 of a heavy chain variable domain of the parent antibody.4. The antibody variant of claim 1 wherein about one to about 30 aminoacid residues have been inserted in or adjacent to the hypervariableregion of the parent antibody.
 5. The antibody variant of claim 4wherein about two to about ten amino acid residues have been inserted inor adjacent to the hypervariable region of the parent antibody.
 6. Theantibody variant of claim 1 which has a binding affinity for saidantigen that is at least about five fold stronger than the bindingaffinity of the parent antibody for said antigen.
 7. The antibodyvariant of claim 1 wherein the antibody variant has a potency in abiological activity assay which is at least about 20 fold greater thanthe potency of the parent antibody in the biological activity assay. 8.The antibody variant of claim 7 wherein the potency of the antibodyvariant in the biological activity assay is at least about 50 foldgreater than the potency of the parent antibody in the biologicalactivity assay.
 9. The antibody variant of claim 1 wherein the parentantibody is a humanized antibody.
 10. The antibody variant of claim 1wherein the parent antibody is a human antibody.
 11. The antibodyvariant of claim 1 wherein at least one of the inserted residues has anet positive charge or a net negative charge.
 12. The antibody variantof claim 11 wherein at least one of the inserted residues is arginine orlysine.
 13. The antibody variant of claim 3 wherein the insertion isadjacent to residue number 100 of the heavy chain variable domain of theparent antibody, utilizing the variable domain residue numbering as inKabat.
 14. The antibody variant of claim 13 wherein the insertionconsists of about three inserted amino acid residues.
 15. The antibodyvariant of claim 1 further comprising an amino acid substitution in thehypervariable region.
 16. The antibody variant of claim 1 whichcomprises a heavy chain variable domain, wherein CDR H3 of a heavy chainvariable domain of the variant antibody comprises the amino acidsequence of SEQ ID NO:85.
 17. The antibody variant of claim 16 whichcomprises a heavy chain variable domain comprising the amino acidsequence in SEQ ID NO:98 or SEQ ID NO:99.
 18. A composition comprisingthe antibody variant of claim 1 and a pharmaceutically acceptablecarrier.
 19. An antibody variant comprising a heavy chain variabledomain, wherein CDR H3 of the heavy chain variable domain comprises theamino acid sequence of CDR H3 of a variant selected from the groupconsisting of Y0239-19 (SEQ ID NO:85); Y0239-8 (SEQ ID NO:53); Y0240-1(SEQ ID NO:86); Y0239-12 (SEQ ID NO:78); Y0239-9 (SEQ ID NO:54); andY0261-6 (SEQ ID NO:89).
 20. A method for producing an antibody variantcomprising introducing an amino acid residue in or adjacent to ahypervariable region of a parent antibody, wherein the antibody varianthas a binding affinity for a target antigen which is at least about twofold stronger than the binding affinity of the parent antibody for saidantigen.
 21. The method of claim 20 wherein the hypervariable region inwhich the amino acid residue is introduced is one which is involved inbinding the antigen in the parent antibody.
 22. A method for making anantibody variant, comprising the steps of: (a) identifying potentialamino acid interactions between a hypervariable region of a parentantibody and a target antigen; (b) preparing a variant of the parentantibody comprising introducing an amino acid residue in or adjacent tothe hypervariable region of the parent antibody, wherein the introducedamino acid residue contributes to the potential amino acid interactionsin (a); and (c) selecting an antibody variant prepared as in (b) whichhas a stronger binding affinity for said antigen than the parentantibody.
 23. The method of claim 22, wherein step (a) involvesanalyzing a molecular model of the parent antibody complexed with saidantigen.
 24. The method of claim 22 wherein step (b) comprises preparingantibody variants displayed on phage.
 25. The method of claim 22 whereinthe amino acid interactions are selected from the group consisting ofhydrogen-bonding, Van der Waals interactions and ionic interactions. 26.Isolated nucleic acid encoding the antibody variant of claim
 1. 27. Avector comprising the nucleic acid of claim
 26. 28. A host celltransformed with the vector of claim
 27. 29. A process of producing anantibody variant comprising culturing the host cell of claim 28 so thatthe nucleic acid is expressed.
 30. The process of claim 29 furthercomprising recovering the antibody variant from the host cell culture.31. The process of claim 30 wherein the antibody variant is recoveredfrom the host cell culture medium.